Cell signaling through O-GlcNAc reader proteins

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Project Summary/Abstract O-linked b-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification (PTM) in mammals, decorating thousands of nuclear and cytoplasmic proteins. O-GlcNAc cycling is an essential regulator of myriad aspects of cell physiology and is dysregulated in numerous human diseases, such as cancer, diabetes and neurodegeneration. Despite this broad pathophysiological significance, major aspects of O-GlcNAc signaling remain incompletely understood, including the biochemical mechanisms through which O-GlcNAc transduces information. At the start of our project, several studies had shown that O-GlcNAcylation mediates protein-protein interactions (PPIs) in processes as diverse as chromatin remodeling, deubiquitination and nuclear envelope assembly. These results suggested that O-GlcNAc might signal through conserved modes of PPI. However, little was known about either the structure or function of these intracellular glycoprotein-protein complexes. We hypothesized that mammalian “reader” proteins might exist and transduce O-GlcNAc signals. In the first project period, we devised and deployed a method to identify proteins that bind specifically and directly to O- GlcNAcylated peptides and proteins in vitro and in human cells. These include the 14-3-3 family, which are well- known to bind to phosphoproteins. Our results indicate that 14-3-3 proteins may be signal integrators, mediating the extensively documented crosstalk between O-GlcNAc and O-phosphate, with broad implications for intracellular signaling. In addition, we identified and characterized O-GlcNAc-mediated PPIs among nuclear import receptors, intermediate filament (IF) cytoskeleton proteins and ubiquitin E3 ligase complex components. Building on these results, we will study a focused set of diverse model O-GlcNAc-binding proteins in the next project period. This strategy will generate new insight into the functions of individual O-GlcNAc-mediated PPIs in particular cell biological processes, while also providing a comparative perspective across reader proteins, allowing us to discern general themes of O-GlcNAc-mediated PPIs at the biophysical, biochemical and physiological levels. The long-term objective of our work is to understand the principles, mechanisms and functions of O- GlcNAc-mediated PPIs in mammals. In the next project period, we will advance this goal through three Specific Aims. In Aim 1, we will define the biochemical scope and phenotypic effects of O-GlcNAc binding by 14-3-3 proteins. In Aim 2, we will determine the role of O-GlcNAc/nuclear transport receptor interactions in inter-organelle trafficking. In Aim 3, we will dissect the functional importance of O-GlcNAc-mediated interactions in two model IF proteins. We believe our proposed work will significantly advance the field of intracellular signaling both by elucidating the mechanisms and functions of individual O-GlcNAc-mediated PPIs and by revealing conserved biochemical principles of O-GlcNAc recognition across reader proteins.